Modified seb and prophylactics/remedies for immunopathy containing the same

ABSTRACT

A novel prophylactic/remedy for immunopathy is provided which is not neutralized by a neutralizing antibody to Staphylococcal enterotoxin B (SEB), known as one of superantigens, and may effectively act as a superantigen. A modified SEB having a reduced reactivity with a neutralizing antibody to SEB (anti-SEB antibody) and a prophylactic/remedy for immunopathy comprising as an active ingredient said modified SEB. The modified SEB of the present invention may be prepared with the evolutionary molecular engineering technique by introducing amino acid substitution in the amino acid sequence of SEB, especially at an epitope recognition site of the anti-SEB antibody in the amino acid sequence of SEB.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to novel prophylactics/remedies forimmunopathy. More specifically, the present invention relates tomodified forms of Staphylococcal enterotoxin B (hereinafter referred toas “SEB”), known as one of superantigens, and prophylactics/remedies forimmunopathy such as rheumatoid arthritis, allergic diseases, etc.comprising as an active ingredient said modified SEB.

BACKGROUND OF THE INVENTION

Autoimmune diseases are classified into two types: organ-nonspecifictype autoimmune diseases such as rheumatoid arthritis (hereinafter alsoreferred to as “RA”) and organ-specific type autoimmune diseases such asulcerative colitis. They are induced by T cells responsive to selfantigens, said T cells being normally under immunological tolerance,that were activated within self tissues by certain causes to respond toself antigens, leading to continuous inflammatory reactions to therebydamage tissues. In such cases, self antigens are type II collagen thatconstitutes self joint or main components of the mucous membrane of theintestine, respectively.

The number of patients suffering from these diseases has been slightlyincreasing year by year but no effective remedies or prophylaxis havebeen found (“Immunodeficiency due to medicament”, Men-eki Kagaku(Immunological Science), Vol. 9. p. 285-289 (1984) Ed. by YuichiYamamura, Chuzo Kishimoto, Robert A. Good). Currently, for treatment ofthese diseases, there have been employed pharmacotherapy includingadministration of Salazopyrin, 5-aminosalycic acid, azathioprine, 6-MP,tranilast, methotrexate, cyclosporine A, or metronidazole, andadministration of an excess amount of 7S-immunoglobulin; surgicaltherapy such as thymectomy or replacement with artificial joint; orsymptomatic therapy such as nutritional therapy (Yoichi Ichikawa et al.“Study on efficacy of long-term administration of methotrexate andsalazosulfapyridine on rheumatoid arthritis case” Rheumatism, 1995, Vol.35, p. 663-670; Sadao Kashiwazaki, “Study on efficacy of combination ofauranofin and methotrexate on rheumatoid arthritis”, Rheumatism, 1996,Vol. 36, p. 528-544; Takefumi Furutani et al., “Detrimental event intherapy with low dose methotrexate on rheumatoid arthritis”, Rheumatism,1996, Vol. 36, p. 746-752; Nobuo Watanabe, “Pharmacotherapy on juvenilerheumatoid arthritis”, Rheumatism, 1996, Vol. 36, p. 670-675; TakayasuYakura, “Immunosuppressive therapy: Treatment of autoimmune diseases”,Sogo Rinsho, 1981, Vol. 30, p. 3358; and Shin Totokawa et al., “Study onmethotrexate therapy in rheumatoid arthritis: Seeking for strategy ofmore effective administration”, Rheumatism, 1997, Vol. 37, p. 681-687).However, these therapies are not eradicative but rather aredisadvantageous in that they may cause severe adverse side effects dueto long-term ingestion of medicaments. Thus, it is desired to developmore effective prophylactics/remedies and therapy.

SEB is one of enterotoxins (causative toxins of toxin-type foodpoisoning) produced by Staphylococcus aureus. SEB consists of 239 aminoacid residues and its amino acid sequence is also known. The SEBmolecule comprises two domains, the first domain consisting of residues1 to 120, and the second domain consisting of residues 127 to 239. Atthe N-terminal of SEB, three Regions, Region 1 consisting of residues 9to 23, Region 2 consisting of residues 41 to 53 and Region 3 consistingof residues 60 to 61, were identified that may affect binding of classII Major Histocompatibility Complex (hereinafter referred to as “MHC”)and/or binding of T cell antigen receptor (hereinafter referred to as“TCR”).

As is well known, SEB is one of bacterial superantigens (White J. etal., Cell, 1989, Vol. 56, p. 27-35). Normal antigens, being complexedwith class II MHC, are recognized by TCR on T cells and this recognitionis restricted to a haplotype of class II MHC molecule, called “MHCrestriction”. On the contrary, superantigens are bound to class II MHCmolecule irrespective of haplotype and further to a specific β chainvariable region (Vβ chain) of TCR. As a consequence, T cells bound withthe superantigen are transiently activated, are promoted to divide andpropagate and produce inflammatory cytokines (Micusan V. V. & ThibodeanJ., Seminars in Immunology, 1993, Vol. 5, p. 3-11).

When a superantigen is intravenously or intraperitoneally administeredto newborn mice, a subpopulation of T cells having VβTCR responsive tothe superantigen is eliminated so that said mice become non-responsiveto said antigen, i.e. immunological tolerance. On the other hand, whenSEB is administered to adult mice, the condition where T cells bearingVβTCR that binds to the superantigen become non-responsive to furtherstimulation with the superantigen, i.e. anergy, is induced, to therebycause immunological tolerance. Such features of a superantigen aredistinct from the normal antigen recognition. With the ability to induceimmunological tolerance in T cells bearing the specific VβTCR, SEB issuggested to be applicable for prevention or treatment of certainimmunopathy, in particular, type I allergic diseases or autoimmunediseases. Indeed, it is reported that SEB administration to a system ofdisease model mice allowed for inhibition of onset of said disease.

Kim C. et al. reported that lupus nephritis in MRL/lpr mice, model miceof Systemic lupus erythematosus (hereinafter referred to as “SLE”),could be suppressed by previously administering SEB (Kim C. et al.,Journal of Experimental Medicine, 1991, vol. 174, p. 1131). Rott O. etal. also reported that SEB was previously administered to a system ofExperimental Allergic Encepharomyelitis (hereinafter referred to as“EAE”) to induce immunological tolerance in T cells bearing the Vβ8TCRresponsive to SEB to thereby suppress the disease (Rott O. et al.,International and National Immunology, 1991, vol. 4, p. 347). Theseresults suggest a possibility that SEB may be used as a vaccine to allowfor prevention of specific autoimmune diseases.

However, in these experiments, SEB was administered intravenously orintraperitoneally with a dose of as much as around 100 μg per animal.With such a high dose, an extent of pathogenicity not disregarded willinevitably be introduced to mice and antigenicity and immunogenicity arealso problematic. In particular, as described above, a superantigen,when administered at a large amount, will induce transient activation ofthe subpopulation of T cells or antigen-presenting cells to inviteacceleration of inflammatory cytokine production, resulting in the acuteinflammatory condition within the living body. Besides, in case ofhuman, Kuwahata et al. reported that an anti-SEB antibody is present inblood from almost 100% of children of more than the school age and ananti-IgA antibody is detected in about 50% of the children from analysisof saliva et al. (M. Kuwahata et al., Acta Pediatrica Japonica, 1996,38, p. 1-7). Origuchi et al. also demonstrated that a level of IgM-typeanti-SEB antibody is significantly high in serum from patients sufferingfrom rheumatism (Origuchi et al., Annals of the Rheumatic Disease 1995,54, p. 713-720). Moreover, Nishi et al. revealed that a major epitope ofan anti-SEB antibody in human serum is located at a C-terminal of SEBand an antibody against said C-terminal region is a neutralizingantibody to SEB (Jun-Ichiro Nishi et al., The journal of Immunology,1997, 158, p. 247-254). This implies that, when SEB is administered tohuman, SEB will be neutralized for its biological activity by theantibody and eliminated from the living body. Thus, Nishi et al.constructed a mutant SEB lacking the major epitope at the C-terminalusing the genetic engineering technique. However, the thus preparedmodified SEB could only be expressed in an insoluble form to hamperthorough assessment and analysis. Also said modified SEB could not copewith stable supply of medicine.

To tackle the various problems in association with administration of alarge amount of a superantigen, the present inventors provided a measurefor effectively inducing immunological tolerance by orally administeringa highly purified SEB in a dose not inducing pathogenicity successivelyfor a long period of time (Japanese Patent Publication No. 110704/1997)and constructed a modified SEB and a derivative thereof throughmolecular alteration of SEB in which inherent toxicity of SEB is reducedwhile its preventive/therapeutic effect for immunopathy is maintained toprove utility of SEB (WO99/40935).

DISCLOSURE OF THE INVENTION Technical Problem to be Solved by theInvention

As described above, there is a problem that, when SEB in a natural formis administered to human, SEB will be neutralized for its biologicalactivity by the anti-SEB antibody occurring in the living body whichultimately eliminates SEB from the living body and hence a desiredeffect of SEB is not expected. Thus, a mutant SEB lacking the majorepitope at the C-terminal has been constructed using the geneticengineering technique but the thus prepared modified SEB could only beexpressed in an insoluble form to hamper thorough assessment andanalysis and could not cope with stable supply of medicine.

Means for Solving the Problems

In order to solve the above problem relating to antigenicity of SEB, thepresent inventors have studied using the evolutionary molecularengineering technique. As a consequence, by introducing amino acidsubstitution into naturally occurring SEB or the known modified SEBs andperforming screening among the resulting modified SEBs, the presentinventors could successfully prepare a modified SEB that has a reducedbinding to the anti-SEB neutralizing antibody and is capable of beingexpressed in a soluble form in E. coli to be maintained stably in anaqueous solution and that retains the therapeutic effect to immunopathyequivalent to that of naturally occurring SEB.

The present invention provides for a modified SEB that has a reducedbinding to a neutralizing antibody to Staphylococcal enterotoxin B (SEB)(anti-SEB antibody).

MORE EFFICACIOUS EFFECTS THAN PRIOR ART

The epitope-modified SEBs of the present invention were demonstrated tohave a reduced reactivity with a neutralizing antibody to SEB and tohave an activity to ameliorate the symptoms of CIA (collagen-inducedarthritis) equivalent to that of N23Y [a mutant in which asparagineresidue at 23-position in SEB is substituted with tyrosine; WO99/40935(PCT/JP99/00638)]. These epitope-modified SEBs are capable of beingexpressed and secreted in a soluble form and expected for use asefficacious prophylactics/remedies for immunopathy such as rheumatoidarthritis, allergic diseases, and the like.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of inhibition test of human PBMC (peripheralblood mononuclear cells) activation by SEB using anti-SEB neutralizingmonoclonal antibody SA58-2.

FIG. 2 shows reactivity of the epitope-modified SEBs of the presentinvention with (A) anti-SEB neutralizing monoclonal antibody SA58-2, or(B) human anti-SEB antibody.

FIG. 3 shows the results of measurement of proliferative response ofcells with counts of tritium-thymidine after stimulation of human PBMCwith the epitope-modified SEBs of the present invention for three days.

FIG. 4 shows a ratio of blast formation (%) as a result of measurementof blastogenic transformation reaction of cells through flow cytometryafter stimulation of human PBMC with the epitope-modified SEBs of thepresent invention for six days.

FIG. 5 shows (B) the results obtained by measurement of variouscytokines secreted into culture supernatant with ELISA after stimulationof human PBMC with the epitope-modified SEBs of the present inventionfor two days, and (A) relative values of said measurement as compared towild-type SEB.

FIG. 6 shows the results of assessment of the epitope-modified SEBs ofthe present invention for inhibition in swelling of limbs in mousecollagen-induced arthritis (CIA) model.

FIG. 7 shows the results of assessment of the epitope-modified SEBs ofthe present invention for inhibition in bone destruction in mousecollagen-induced arthritis model.

FIG. 8 shows the measurements (absorbance at 450 nm) of (A) anti-SEBantibody titer and (B) anti-47-C7 antibody titer in blood of mice whenthe epitope-modified SEBs of the present invention are orallyadministered.

BEST MODE FOR CARRYING OUT THE INVENTION

The modified SEBs of the present invention may be obtained byintroducing arbitrary amino acid substitution into the amino acidsequence of SEB so that it has a reduced reactivity with an anti-SEBantibody. Such introduction of amino acid substitution may preferably bedone at an epitope recognition site of an anti-SEB antibody in the aminoacid sequence of SEB.

The most preferable site for amino acid substitution in accordance withthe present invention is a region from Lys at 226-position to Lys at229-position in the amino acid sequence of SEB (SEQ ID NO: 1).

The modified SEBs having a reduced reactivity with an anti-SEB antibodyin accordance with the present invention include those having any of thefollowing amino acid sequences for the amino acid sequence from theresidue at 226-position to the residue at 229-position in the amino acidsequence of SEB: (1) Leu Phe Ala Ala; (SEQ ID NO: 2) (2) Ala Thr ThrGln; (SEQ ID NO: 3) (3) Lys Arg Ile Ile. (SEQ ID NO: 4)

The modified SEBs in accordance with the present invention also includethose having substitution of Asn at 23-position in the amino acidsequence of SEB with Tyr in combination with the amino acidsubstitutions as described above.

The present invention further provides for prophylactics/remedies forimmunopathy comprising as an active ingredient the modified SEB obtainedin accordance with the present invention wherein saidprophylactics/remedies has a reduced immunological response to SEB andan inhibitory activity to T cell activation. Immunopathy includes, forinstance, rheumatoid arthritis, allergic diseases, and similar diseases.

The “evolutionary molecular engineering technique” used for preparingthe modified SEB of the present invention is an approach in which anatural process of evolution (natural selection) in living organisms (itis believed that the probability that an amino acid in a certain proteinis substituted with another amino acid is only around once per 10⁷years) is artificially drastically accelerated in vitro for the purposeof designing useful proteins etc. to allow for evolution (acquisition ofnovel function, improvement in function, etc.) to occur in severalmonths, which usually takes several ten thousands years in the naturalworld.

In accordance with the present invention, for preparing the modified SEBhaving a reduced binding to an anti-SEB neutralizing antibody, aminoacid substitution was randomly introduced into the epitope region in SEBrecognized by an anti-SEB neutralizing antibody and the resultingmodified SEBs were screened for those having a reduced binding to ananti-SEB neutralizing antibody. As described above, it is known that themajor epitope of SEB is located at the C-terminal (225 to234-positions). Within this region, the present inventors had attemptedto perform amino acid substitution in four amino acid residues at 226-to 229-positions in the amino acid sequence of SEB. Thus, modified SEBswere prepared and used as a population for selection in vitro in whichfour amino acid residues at 226- to 229-positions in the amino acidsequence of SEB were arbitrarily substituted with any of the naturallyoccurring 20 amino acids. From the population, modified SEBs having areduced binding to an anti-SEB neutralizing antibody were screened. Thescreened modified SEBs were also confirmed that they are capable ofbeing expressed in s soluble form in E. coli to be maintained stably inan aqueous solution and retain the therapeutic effect to immunopathyequivalent to that of naturally occurring SEB.

Specifically, the modified SEBs of the present invention wereconstructed using the phage display technique as described below.

(1) Construction of Phage Display Library of Wild-Type SEB andIdentification of Antigenicity of SEB

M13 phages are initially constructed that display wild-type SEB. Using awild-type SEB expression plasmid incorporated into an expression vector,e.g. pTrc99A (Amersham-Pharmacia), as a template, PCR (polymerase chainreaction) amplification is performed with 5′ and 3′ primers in whichSfiI or NotI recognition sequence is added, respectively. The amplifiedproducts are digested with the restriction enzymes SfiI-NotI andincorporated into a plasmid. E. coli is then transformed with saidplasmid and infected with helper phages to allow for expression of phageparticles that display wild-type SEB as a fusion protein with phage g3protein (SEB-g3 fusion protein). The expression may be confirmed byWestern blotting using anti-SEB rabbit polyclonal antibody.

The phages that display SEB are then serially diluted and measured fortheir reactivity with an anti-SEB neutralizing monoclonal antibody, ananti-SEB antibody derived from human plasma and an anti-Etag antibody inELISA to determine if SEB is displayed on the surface of the phages withits antigenicity being retained.

(2) Construction of Random Mutant Phage Display Library

Random mutant phage display library is constructed in which randommutations are introduced into SEB or the known modified SEBs at 226- to231-positions at the C-terminal. Using a plasmid in which SEB or theknown modified SEBs are incorporated into an expression vector as atemplate, PCR is performed to introduce random mutations at 226- to231-positions. For the known modified SEBs, N23Y [WO99/40935(PCT/JP99/00638)], for instance, may be used which is a mutant withsubstitution of the asparagine residue at 23-position of SEB withtyrosine residue.

Mutation may be introduced, for instance, as described below. SEB randomgenes for arbitrary expression of any of twenty amino acids at 226- to229-positions are prepared using 5′ primer in which SfiI recognitionsequence, corresponding to the N-terminal of the full-length SEB, isadded and 3′ primer in which NotI recognition sequence is added withboth primers being incorporated with four repeats of NNK (N is any of A,C, G or T; K is G or T) at the codons corresponding to each amino acidat 226- to 229-positions. After treatment with SfiI/NotI, the SEB randomgenes are incorporated into plasmids and E. coli is transformed withsaid plasmids to construct a mutant library.

This transformant library is then infected with helper phages to allowfor expression of phage particles that display SEB random as a fusionprotein with phage g3 protein.

(3) Screening of Modified SEBs Having Low Reactivity with Anti-SEBNeutralizing Antibody

The library described above is screened for the reactivity with ananti-SEB neutralizing monoclonal antibody and efficiency of Etagexpression. For the anti-SEB neutralizing monoclonal antibody, SA58-2(manufactured by Juridical Foundation The Chemo-Sero-TherapeuticResearch Institute), for instance, may be used as its neutralizingactivity has been confirmed. Specifically, in the first step, the randommutant phage display library is reacted with a plate with an immobilizedanti-Etag antibody to select phages that express the fusion protein ofthe modified SEB (g3-Etag-modified SEB) in a soluble form. In the secondstep, the phages selected in the first step are further reacted with aplate with an immobilized anti-SEB neutralizing monoclonal antibody tothereby recover phages that are incapable of reacting with the antibody.The obtained clones are isolated and analyzed for the sequence of theepitope region where the mutation is introduced, expression of the g3fusion protein, a site of expression, and reactivity with a humananti-SEB antibody.

(4) Selection and Assessment of Modified SEBs Having Low Reactivity withAnti-Seb Neutralizing Antibody

The clones obtained in step (3) above are further screened into severalclones with indices of expression in a soluble form, an expression leveland reactivity with an anti-SEB antibody. The obtained clones areassessed for their reactivity with an anti-SEB monoclonal antibody andan affinity-purified human anti-SEB antibody in sandwich ELISA.

Finally, the amino acid sequence at the epitope region in the cloneshaving low reactivity is determined.

The present invention is illustrated in more detail by means of thefollowing Examples but should not be construed to be limited thereto.

EXAMPLE 1

Inhibition of T Cell Activation by SEB with Anti-SEB Antibody

Using SA58-2 antibody (manufactured by Juridical Foundation TheChemo-Sero-Therapeutic Research Institute), a neutralizing monoclonalantibody with specificity against SEB, and JF2 antibody (manufactured byJuridical Foundation The Chemo-Sero-Therapeutic Research Institute) withspecificity against Japanese encephalitis virus, SA58-2 antibody wasassessed for its ability to neutralize SEB.

Peripheral blood mononuclear cells from healthy adults were inoculatedto a 96-well plate at 1×10⁵ cells/well. The cells were stimulated forthree days with SEB (Toxin Technology, Inc.) added to the plate at aconcentration of 1 ng/mL and simultaneously SA58-2 and JF2 antibodieswere added to the plate at 0.05, 0.5, 5 or 50 ng/mL. Sixteen hoursbefore harvest, the cells were allowed to take up tritium-thymidine (0.5μCi) for investigating the proliferation-inducing activity.

As a result, the proliferation-stimulating effect by SEB was notinhibited when 50 ng/mL of JF2 antibody was added. On the other hand,when SA58-2 antibody was added, an inhibitory effect of not less than80% was detected with addition of 5 ng/mL or more (FIG. 1). Thus, it wasproved that SA58-2 antibody was a neutralizing antibody that maysufficiently inhibit the lymphocyte-activating capacity of SEB.

EXAMPLE 2

Reactivity of Modified SEBs with Anti-SEB Antibody

(1) Construction of Phage Display Library of Wild-Type SEB andIdentification of Antigenicity of SEB

Construction of the modified SEBs having low reactivity with an anti-SEBantibody was performed as described below using the evolutionarymolecular engineering technique. M13 phages were initially constructedthat display wild-type SEB. Using a wild-type SEB expression plasmid(pTrc99A/SEB) incorporated into an expression vector pTrc99A(Amersham-Pharmacia) as a template, PCR (polymerase chain reaction)amplification was performed with 5′ and 3′ primers in which SfiI or NotIrecognition sequence was added, respectively. The amplified productswere digested with the restriction enzymes SfiI-NotI and incorporatedinto pCANTAB5E (Pharmacia). This plasmid was referred to as “pCAN/SEB”.E. coli TG1 was then transformed with pCAN/SEB and infected with helperphages to allow for expression of phage particles that display wild-typeSEB as a fusion protein with phage g3 protein (SEB-g3 fusion protein).The expression was confirmed by Western blotting using an anti-SEBrabbit polyclonal antibody.

The phages that display SEB were then serially diluted and measured fortheir reactivity with the anti-SEB neutralizing monoclonal antibodySA58-2 (manufactured by Juridical Foundation The Chemo-Sero-TherapeuticResearch Institute), anti-SEB antibody derived from human plasma andanti-Etag antibody (Amersham-Pharmacia) in ELISA to determine if SEB wasdisplayed on the surface of the phages with its antigenicity beingretained. The phages that display SEB were serially diluted by ten-foldstarting from 1×10¹⁰ and added to a 96-well plate. The plate was reactedwith the anti-SEB antibodies or the anti-Etag antibody and the reactionwas developed with HRP-labeled secondary antibody and absorbance wasdetermined. As a result, it was proved that SEB was expressed andretained on the surface of the phages with its antigenicity equivalentto that of naturally occurring SEB.

(2) Construction of Random Mutant Phage Display Library

Using as a template a plasmid pTrc99A/N23Y in which one of the knownmodified SEBs, N23Y [a mutant with substitution of the asparagineresidue at 23-position of SEB with tyrosine residue: WO99/40935(PCT/JP99/00638)] was incorporated into pTrc99A, PCR was performed tointroduce random mutations at 226- to 229-positions. Mutation wasintroduced as described below.

N23Y random genes for arbitrary expression of any of twenty amino acidsat 226- to 229-positions were prepared using 5′ primer in which SfiIrecognition sequence, corresponding to the N-terminal of the full-lengthSEB, was added and 3′ primer in which NotI recognition sequence wasadded with both primers being incorporated with four repeats of NNK (Nis any of A, C, G or T; K is G or T) at the codons corresponding to eachamino acid at 226- to 229-positions. After treatment with SfiI/NotI, theN23Y random genes were incorporated into pCANTAB5E. This plasmid wasreferred to as “pCAN/N23Yrandom”. E. coli TG1 was transformed withpCAN/N23Yrandom to thereby construct a library comprising 1.88×10⁵mutants.

This transformant library was then infected with helper phages to allowfor expression of phage particles that displayed N23Y random as a fusionprotein with phage g3 protein. Reactivity of the phage library withanti-Etag antibody, SA58-2 antibody, and affinity-purified humananti-SEB antibody was analyzed in ELISA. As a result, it was confirmedthat a rate of display on the phage of N23Y random was only 1/20 of thatof wild-type SEB but its reactivity with SA58-2 antibody and with theaffinity-purified human anti-SEB antibody was much reduced, i.e. as lowas 1/200 or less of that of wild-type SEB. Accordingly, it was estimatedthat the reactivity of N23Y random consisting of the library with eachof the antibodies was reduced by about 1/10 on an average as compared tothat of wild-type SEB, which implied that there were indeed present inthis library the sequences having low reactivity with these anti-SEBantibodies.

(3) Screening of Modified SEBs Having Low Reactivity with Anti-SEBNeutralizing Antibody

The library described above was screened thrice for the reactivity withSA58-2, i.e. the anti-SEB neutralizing monoclonal antibody with itsneutralizing activity being confirmed, and efficiency of Etagexpression. Thus, in the first step, the random mutant phage displaylibrary was reacted with a plate with an immobilized anti-Etag antibodyto select phages that express the fusion protein of the modified SEB(g3-Etag-modified SEB) in a soluble form. In the second step, the phagesselected in the first step were further reacted with a plate with theimmobilized anti-SEB neutralizing monoclonal antibody SA58-2 to therebyrecover phages that were incapable of reacting with the antibody. Amongthese, 48 clones were arbitrarily isolated and analyzed for the sequenceof the epitope region where the mutation was introduced, expression ofthe g3 fusion protein, a site of expression, and reactivity with a humananti-SEB antibody.

As a result, 30 among these 48 clones were expressed as a fusion proteinwith g3 and 21 among these 30 clones were capable of being expressed ina culture supernatant. Among these 21 clones, 10 clones were evidentlyreactive with a human anti-SEB antibody while the remaining 11 clonesexhibited extremely lowered reactivity.

(4) Selection and Assessment of Modified SEBs Having Low Reactivity withAnti-SEB Neutralizing Antibody

Analysis was further continued for the clones with indices of expressionin a soluble form, an expression level and reactivity with anti-SEBantibody to select eight clones; 4-C1, 4-C3, 42-C2, 42-C3, 47-C3, 47-C7,48-C1 and 48-C4. These clones were assessed for their reactivity withSA58-2 antibody and an affinity-purified human anti-SEB antibody insandwich ELISA.

The reactivity of these clones with both antibodies was assessed, takentogether with their expression level. As a result, the reactivity wasreduced in all of these clones (FIG. 2). In particular, the reactivitywith the neutralizing antibody SA58-2 was reduced by about 1/30 to 1/50in the clones 42-C2, 48-C1 and 48-C4, by about ⅛ in 47-C7, and by about½ in 4-C1, as compared to N23Y used as a template (Table 1). As for thereactivity with the purified human anti-SEB antibody, it was reduced byabout ⅛ in 42-C2, 48-C1, 48-C4 and 47-C7, and by about ½ to ¼ in 4-C1(Table 1). For these clones in which their reactivity with theseanti-SEB antibodies was assessed, each of the amino acid sequences atthe epitope region was also determined and shown in Table 1.

By assessing the reactivity as described above as a whole, it wasdetermined that 42-C2, 47-C7 and 4-C1 were used in the subsequentexperiments. These modified SEBs, obtained with N23Y as a template, arehereinafter collectively referred to as “epitope-modified SEBs”. TABLE 1Reduced reactivity of epitope-modified SEBs with anti-SEB antibodyReactivity Reactivity Clone Sequence of epitope with anti- with humanNos. region SEB mAb anti-SEB Ab N23Y SKDVKIEVYL 1 1 (SEQ ID NO: 5) 42-C2SLFAAIEVYL   1/50 ⅛ (SEQ ID NO: 6) 47-C7 SATTQIEVYL ⅛ ⅛ (SEQ ID NO: 7)4-C1 SKRIIIEVYL ½ ½ to ¼ (SEQ ID NO: 8) 48-C4 SPQPDIEVYL   1/30 ⅛ (SEQID NO: 9)

EXAMPLE 3

Analysis of Biological Activity of Epitope-Modified SEBs to PeripheralBlood Mononuclear Cell

(1) Assessment of Epitope-Modified SEBs for their Proliferation-Inducingor Blast Formation-Inducing Activity

Peripheral blood mononuclear cells (hereinafter also referred to as“PBMC”) from healthy adults were inoculated to a 96-well plate at 1×10⁵cells/well. SEB, N23Y and the epitope-modified SEBs were added to theplate at a concentration of 0.01, 1, 100 and 1000 ng/mL to stimulate thecells for three days. Sixteen hours before harvest, the cells wereallowed to take up tritium-thymidine (0.5 μCi) for investigating theproliferation-inducing activity. Also, the above PBMC were cultured inthe presence of the modified SEBs at the same concentration for a mediumperiod of time (6 days) to investigate an extent of blast formation of Tcells by FSC/SSC analysis of flow cytometry (hereinafter also referredto as “FACS”).

As a result, SEB exhibited a potent proliferation-inducing activity toPBMC at 0.01 ng/mL or more in a concentration dependent manner. Typicalexamples are shown in FIG. 3. N23Y had an extremely lowerproliferation-inducing activity than SEB in which an uptake oftritium-thymidine began to be detected at 100 ng/mL or more and wascounted as low as about 1/10 of that of SEB even at 1000 ng/mL. Theepitope-modified SEBs had a further lowered proliferation-inducingactivity (FIG. 3). As for the blast formation-inducing activity, N23Yinduced significant blast formation to 10-30% of the cells at 1 ng/mL ormore while 42-C2 and 47-C7 had about 1/10 of the inducing activity shownby N23Y. 4-C1 exhibited the activity almost equivalent to that of N23Y(FIG. 3). These results revealed that the epitope-modified SEBs, even ifmutation has been introduced into the epitope region, had the biologicalactivity roughly equivalent to N23Y with respect to theproliferation-inducing or blast formation-inducing activity to PBMC invitro.

(2) Assessment of Epitope-Modified SEBs for their Cytokine-InducingActivity

PBMC from healthy adults were inoculated to a 24-well plate at 1×10⁶cells/well. SEB, N23Y and the epitope-modified SEBs were added to theplate at a concentration of 0.01, 1, 100 and 1000 ng/mL to stimulate thecells for two days and a culture supernatant was collected. The culturesupernatant was measured for production of various cytokines (TNF-α,IL-β, IL-6, IL-8, IL-12, IFN-γ, IL-1ra, IL-4, IL-10, GM-CSF) with ELISAkit (CytoSets CytoFix, Asahi Techno Glass Corporation).

As a result, the epitope-modified SEBs like N23Y had a lower activity toproduce cytokines than SEB with a similar cytokine-inducing pattern tothat of N23Y. Specifically, they produced inhibitory cytokines such asIL-1ra, IL-10 and IL-4 at a relatively significantly higher level thanSEB while they induced inflammatory cytokines such as IL-1β, IL-6,TNF-α, IL-12, GM-CSF and IFN-γ at a relatively lower level than SEB.FIG. 5 shows a relative activity of the epitope-modified SEBs (100ng/mL) as compared to SEB with the cytokine-inducing activity of SEB at100 ng/mL being 100%. The epitope-modified SEBs had the biologicalactivity equivalent to that of N23Y with respect to thecytokine-inducing activity to PBMC in vitro.

Besides, since N23Y and the epitope-modified SEBs exhibited a remarkablyrestricted induction of IFN-γ but a relatively higher production of theinhibitory cytokines IL-4 and IL-10 as compared to SEB as clearly shownin the results of FIG. 5, it was supposed that the modified SEBs had anactivity to shift T populations from Th1 to Th2.

EXAMPLE 4

(1) Assessment of Epitope-Modified SEBs in Mouse Arthritis Model

Efficacy of N23Y and the epitope-modified SEBs was assessed in mousecollagen-induced arthritis (CIA) model. DBA/1J male mice of 7 weeks oldwere sensitized with 100 μg/mouse of bovine type II collagen at the rootof tail using Freund's complete adjuvant (FCA). After 3 weeks, the micereceived booster administration of the same antigen to induce arthritis.One week after the booster administration, each mouse was observed fortheir limbs and severity of arthritis was scored. Scoring was made foreach limb with the following criteria: no onset of disease: 0; swellingin one finger: 1; swelling in two to four fingers or swelling in theinstep of limb: 2; swelling in all the fingers or severe swelling: 3. Atotal score of the four limbs was calculated (maximum 12) and was usedas an arthritis score of mouse. A group of mice with slight arthritishaving arthritis score of 1 to 4 was selected and orally administeredwith the chemical. The chemical was administered using a probe at 10μg/mouse every day for four weeks. After initiation of the chemicaladministration, mice were scored twice a week and severity of arthritiswas observed. After the administration was completed, the joints of thefour limbs were photographed with soft X-ray and severity of erosion inbone was scored so as to assess the activity to bone destruction.

As a result, the epitope-modified SEBs, 4-C1 and 47-C7, significantlyinhibited the symptoms of arthritis as compared to the control groupwhere saline was administered. In 42-C2, no inhibitory activity was seen(FIG. 6). In case of 47-C7 and 4-C1, the activity to inhibit bonedestruction was also observed (FIG. 7).

(2) Induction of Anti-SEB Antibody by Administration of Epitope-ModifiedSEBs

After completion of the tests, whole blood was taken out and a titer ofanti-SEB antibody in blood was measured by ELISA. As a result, the groupof 47-C7 administration exhibited the titer equivalent to that of thegroup of no chemical administration or the control group of salineadministration and hence had a reduced immunogenicity (FIG. 8 (A)). Inaddition, the modified SEB, 47-C7, was also less inclined to induce anantibody to this protein per se and hence it was assumed that thepossibility was low that the modified epitope sequence became anotherantigenic epitope (FIG. 8 (B)).

1. A modified Staphylococcal enterotoxin B (SEB) having a reducedreactivity with a neutralizing antibody to SEB (anti-SEB antibody). 2.The modified SEB of claim 1 wherein the reactivity with the anti-SEBantibody was reduced by introducing arbitrary amino acid substitution inthe amino acid sequence of SEB.
 3. The modified SEB of claim 2 whereinthe amino acid substitution was introduced at an epitope recognitionsite of the anti-SEB antibody in the amino acid sequence of SEB.
 4. Themodified SEB of claim 2 wherein the amino acid substitution wasintroduced within a region from Lys at 226-position to Lys at229-position in the amino acid sequence of SEB.
 5. The modified SEB ofclaim 4 wherein the amino acid sequence of from 226-position to229-position in the amino acid sequence of SEB is Leu Phe Ala Ala. 6.The modified SEB of claim 4 wherein the amino acid sequence of from226-position to 229-position in the amino acid sequence of SEB is AlaThr Thr Gln.
 7. The modified SEB of claim 4 wherein the amino acidsequence of from 226-position to 229-position in the amino acid sequenceof SEB is Lys Arg Ile Ile.
 8. The modified SEB of claim 1 wherein Asn at23-position in the amino acid sequence of SEB is substituted with Tyr.9. A prophylactic/remedy adapted for immunopathy comprising as an activeingredient the modified SEB as set forth in claim 1, wherein saidprophylactic/remedy has a reduced immunological response to SEB and aninhibitory activity to T cell activation.
 10. The prophylactic/remedyfor immunopathy of claim 9 wherein said immunopathy is rheumatoidarthritis.
 11. The prophylactic/remedy for immunopathy of claim 9 whichis in a form for oral administration.
 12. The modified SEB of claim 2wherein Asn at 23-position in the amino acid sequence of SEB issubstituted with Tyr.
 13. The modified SEB of claim 3 wherein Asn at23-position in the amino acid sequence of SEB is substituted with Tyr.14. The modified SEB of claim 4 wherein Asn at 23-position in the aminoacid sequence of SEB is substituted with Tyr.
 15. The modified SEB ofclaim 5 wherein Asn at 23-position in the amino acid sequence of SEB issubstituted with Tyr.
 16. The modified SEB of claim 6 wherein Asn at23-position in the amino acid sequence of SEB is substituted with Tyr.17. The modified SEB of claim 7 wherein Asn at 23-position in the aminoacid sequence of SEB is substituted with Tyr.
 18. A prophylactic/remedyadapted for immunopathy comprising as an active ingredient the modifiedSEB as set forth in claim 8, wherein said prophylactic/remedy has areduced immunological response to SEB and an inhibitory activity to Tcell activation.
 19. The prophylactic/remedy for immunopathy of claim 18which is in a form for oral administration.
 20. The prophylactic/remedyfor immunopathy of claim 18 wherein said immunopathy is rheumatoidarthritis.